Abstract
Calcium signal propagation from endoplasmic reticulum (ER) to mitochondria regulates a multitude of mitochondrial and cell functions, including oxidative ATP production and cell fate decisions. Ca(2+) transfer is optimal at the ER-mitochondrial contacts, where inositol 1,4,5-trisphosphate (IP(3)) receptors (IP3R) can locally expose the mitochondrial Ca(2+) uniporter (mtCU) to high [Ca(2+)] nanodomains. The Ca(2+) loading state of the ER (Ca(2 + )(ER)) can vary broadly in physiological and pathological scenarios, however, the correlation between Ca(2 + )(ER) and the local Ca(2+) transfer is unclear. Here, we studied IP(3)-induced Ca(2+) transfer to mitochondria at different Ca(2 + )(ER) in intact and permeabilized RBL-2H3 cells via fluorescence measurements of cytoplasmic [Ca(2+)] ([Ca(2+)](c)) and mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)). Preincubation of intact cells in high versus low extracellular [Ca(2+)] caused disproportionally greater increase in [Ca(2+)](m) than [Ca(2+)](c) responses to IP(3)-mobilizing agonist. Increasing Ca(2 + )(ER) by small Ca(2+) boluses in suspensions of permeabilized cells supralinearly enhanced the mitochondrial Ca(2+) uptake from IP(3)-induced Ca(2+) release. The IP(3)-induced local [Ca(2+)] spikes exposing the mitochondrial surface measured using a genetically targeted sensor appeared to linearly correlate with Ca(2 + )(ER), indicating that amplification happened in the mitochondria. Indeed, overexpression of an EF-hand deficient mutant of the mtCU gatekeeper MICU1 reduced the cooperativity of mitochondrial Ca(2+) uptake. Interestingly, the IP(3)-induced [Ca(2+)](m) signal plateaued at high Ca(2 + )(ER), indicating activation of a matrix Ca(2+) binding/chelating species. Mitochondria thus seem to maintain a "working [Ca(2+)](m) range" via a low-affinity and high-capacity buffer species, and the ER loading steeply enhances the IP3R-linked [Ca(2+)](m) signals in this working range.